Digital breast tomosynthesis versus X-ray of the breast specimen for intraoperative margin assessment: A randomized trial

Background Involved resection margins after breast conserving surgery (BCS) often require a re-operation with increased patient anxiety and risk of impaired cosmesis. We investigated the number of re-operations due to involved resection margins after BCS comparing digital breast tomosynthesis(DBT) with X-ray for intraoperative margin evaluation. Furthermore, we assessed the diagnostic accuracy of these methods to predict histopathological margin status. Finally, we evaluated risk factors for re-operation. Methods In this randomized, non-blinded study, 250 invasive breast cancer patients were randomized (1:1), whereof 241 were analyzed intraoperatively with either DBT (intervention, n = 119) or X-ray (standard, n = 122). Pearson's chi-squared test, Fisher's exact test, t-test, logistic and ordinal regression analysis was used as appropriate. Results No difference was found in the number of re-operations between the DBT and X-ray group (16.8 % vs 19.7 %, p = 0.57), or in diagnostic accuracy to predict histopathological margin status (77.5 %, CI: 68.6–84.9 %) and (67.3 %, CI: 57.7–75.9 %), respectively. We evaluated 5 potential risk factors for re-operation: Ductal carcinoma in situ (DCIS) outside tumor, OR = 9.4 (CI: 4.3–20.6, p < 0.001); high mammographic breast density, OR = 6.1 (CI: 1.0–38.1, p = 0.047); non-evaluable margins on imaging, OR = 3.8 (CI: 1.3–10.8, p = 0.016); neoadjuvant chemotherapy, OR = 3.0 (CI: 1.0–8.8, p = 0.048); and T2 tumor-size, OR = 2.6 (CI: 1.0–6.4, p = 0.045). Conclusions No difference was found in the number of re-operations or in diagnostic accuracy to predict histopathological margin status between DBT and X-ray groups. DCIS outside the tumor showed the highest risk of re-operation. Intraoperative methods with improved visualization of DCIS are needed to obtain tumor free margins in BCS.


Introduction
Breast conserving surgery (BCS) followed by whole breast irradiation has become the standard treatment of choice in early invasive breast cancer [1].A recent meta-analysis showed a better survival of BCS with whole breast irradiation compared to mastectomy [2].The aim of BCS is to remove the breast cancer with a sufficient surrounding breast tissue to allow for both tumor-free resection margins, and achievement of an acceptable cosmetic result.The reported percentage of patients that receive one or more additional surgeries (re-operations) due to involved margins with invasive cancer or ductal carcinoma in situ (DCIS) after BCS varies, within a range of 1-37 % [3][4][5][6][7].A re-operation may cause additional anxiety for the patient [8], impair the aesthetic outcome [9], and increase costs for the health system [10].To improve intraoperative evaluation of the resected margins, several tools have been developed.Of these, intraoperative frozen section analysis has shown to be the most accurate [7,11], however it is a resource demanding method and requires immediate assistance from a pathologist.At our department, we use a single X-ray image of the surgical specimen as the standard imaging method for intraoperative margin assessment followed by a post-operative gross assessment of the specimen.We have recently conducted a meta-analysis where X-ray of the breast specimen was the most frequently reported radiological method for intraoperative margin assessment [12], but with a lower diagnostic accuracy in predicting histopathological margin status of 50-69 %, compared to other radiological modalities: Ultrasound 71-87 %, [4,[13][14][15]; micro-CT 62-80 % [16][17][18][19], and digital breast tomosynthesis (DBT) of 67-83 %, [12,[20][21][22][23][24].The DBT creates multiple 1 mm images reconstructed to a series of 2D images that gives a 3D impression of the whole breast specimen.This reduces the risk of breast tissue superimposition with a clearer depiction of the resected margins.We hypothesized that DBT gives a better view of all resected margins during BCS than a single X-ray image resulting in less patients with involved histopathological margins that require a re-operation.
We aimed to evaluate the number of re-operations due to involved resection margins after BCS in a randomized setting, comparing use of DBT with X-ray for intraoperative margin evaluation.Furthermore, we assessed the diagnostic accuracy of these methods to predict histopathological margin status.Finally, we evaluated potential clinicalpathological factors associated with increased risk of a re-operation.

Study population
The study was registered at ClinicalTrials.gov(NCT04478669), approved by the Ethics Committee for Central Region Denmark, and the Danish Data Protection Agency prior to inclusion and reported according to the consolidated standards of reporting trials (CONSORT) [25].Fig. 1.Flowchart of the included study patients according to the consolidated standards of reporting trial (CONSORT) Abbreviations: n, number of patients; DCIS, ductal carcinoma in situ; DBT, digital breast tomosynthesis; MRI, magnetic resonance imaging.*Non-surgical treatment: Breast cancer patients with distant metastases that received chemotherapy and patients that received hormone therapy only.

Study design
We conducted a parallel, randomized, non-blinded, controlled study, where patients were randomized 1:1 to intraoperative DBT (intervention) or X-ray (standard method) of their breast specimen.The randomization was performed in the Research Electronic Data Capture system (REDCap) [26].The random allocation sequences were generated a priori by the independent trial REDCap administrator at Aarhus University.The allocation result was reported in the medical record by the first author, prior to initial BCS.

Eligibility criteria
We included women, age ≥18 years, with biopsy-verified, invasive breast cancer planned for BCS.Inclusion of the first patient was September 17th, 2020, and last patient included, January 28th, 2022, at the Department of Plastic and Breast Surgery, Aarhus University Hospital.Patients planned for neoadjuvant chemotherapy (NACT) were included after completion of their systemic treatment.Patients with previous breast surgery, ductal carcinoma in situ (DCIS) with no invasive cancer, and patients planned for mastectomy were excluded (Fig. 1).

Perioperative data collection
Preoperative sonographic tumor size, mammographic breast density (categorized A-D, A: Almost entirely fatty and D: Extreme dense breast tissue) [27], age, menopausal status, NACT-status, tumor palpability, surgical outcomes (number and type of re-operations), histological tumor size, malignancy grade, histological subtype, DCIS outside the tumor (in the surrounding tissue at a distance from the tumor), human epidermal growth factor 2 (HER-2), estrogen receptor (ER), and lymph node status were collected from medical records and registered in REDCap.

Preoperative marking of the tumor
Patients with a non-palpable breast tumor or where the tumor was not definitely palpable, and palpable tumors of patients planned for NACT had the lesion marked with a coil guided by ultrasound, performed by the breast radiologist and its location visualized on a mammography.
In the morning, prior to surgery, the location of the coil was visualized on mammography and ultrasound to ensure its location and marked with ink on the skin by the breast radiologist.The surgeon used the ink on the patient's skin in combination with ultrasound as guidance Fig. 2. Intraoperative digital breast tomosynthesis of a breast cancer specimen with a true negative finding.a) The operating theater with the digital breast tomosynthesis (DBT), (intervention method) and the X-ray device (standard method).b) Breast specimen from a patient with invasive ductal breast cancer with a true negative finding.c) A sectional DBT-image through the middle and d) bottom of the specimen shows a sufficient distance from the centrally located tumor (white arrow), to the resected margins of 6 mm at 6 o'clock (red line).The radiologist assessed the intraoperative margins as tumor free that was confirmed by the pathologist in the final histopathology.(For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)for excision of the breast tumor.

Intraoperative imaging
After excision of the breast tumor, the surgeon marked the specimen with sutures and clips for orientation and placed it on a styrofoam bed, oriented clockwise and inserted in either the DBT or the X-ray device located in the operating theater (Fig. 2 a), as according to randomization.The Mozart® Specimen Tomosynthesis Imaging System, (KUBTEC Medical Imaging, Stratford, CT, USA) was used for the DBT images and the Faxitron™ intraoperative specimen digital radiography system (Hologic® Inc., Bedford, MA, USA) was used for the X-ray images.The images were sent digitally and analyzed by dedicated breast radiologists, who subsequently communicated their conclusion on margin status to the surgeon over the telephone.Based on the conclusion of the radiologist, a re-resection was performed in case of involvement with tumor or microcalcifications close to or in the resected margins.The surgeon then attached the re-resection to the main specimen before it was sent to pathological examination.Weight of the specimen and total analysis time was registered intraoperatively.Size and visibility of the tumor, overlying skin in the specimen, and if the resected margins were assessed as involved or tumor free was noted on a predefined sheet by the breast radiologists.

Postoperative histopathological analysis
As part of the routine diagnostics, breast pathologists assessed the specimen macro-and microscopically.An involved margin was defined as a margin width of 0 mm for invasive carcinoma and ≤2 mm for DCIS, and tumor free as > 0 mm for invasive cancer, and >2 mm for DCIS according to national and international guidelines [28,29].The histopathological assessment of the resected margins was used as reference for the radiological assessment of the resected margins.
Conclusions from the radiologist based on the allocated imaging method were true positive (TP), if margins were truly involved by invasive cancer, DCIS, or both, and true negative (TN) if margins were tumor free on imaging as well as on final histopathology.False positive (FP) findings corresponded to margins assessed as involved by imaging but microscopically tumor free and false negative (FN), if considered tumor free on imaging but involved on final histopathology.

Statistical analysis
All analyses were performed in Stata 17 (StataCorp, 2021, college station, Texas, USA) with a significance level of 0.05.Prior to inclusion, a sample size calculation showed that 250 patients were needed to detect a reduction of involved margins from 20 % to 7 % between the DBT and X-ray group with a statistical power of 80 %.Patient characteristics were examined for differences between groups with t-test of the means for continuous variables after log-transformation when appropriate.Pearson's chi-squared test and Fisher's exact test (expected count <5) were used for categorical variables.Logistic regressions and ordinal logistic regressions were used to assess differences of the categorical variables between the groups with and without adjustment for possible confounders.Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy of DBT and X-ray were calculated from raw diagnostic accuracy data.Significant risk factors for re-operation were examined by logistic regressions with and without adjustment for confounders, and with Bonferroni correction for multiple testing in the pairwise comparisons of breast density categories.

Study demographics
A total of 250 invasive breast cancer patients were included and randomized.Nine out of 250 patients (6 with DBT and 3 with X-ray) were excluded due to eligibility criteria (Fig. 1).Subsequent analysis encompassed 241 breast specimens from 241 breast cancer patients, 119 with DBT (intervention) and 122 with standard X-ray.The perioperative patient characteristics between the DBT and X-ray groups were comparable (Table 1).

Final histopathological margin status and surgical outcomes
There was no difference in the percentage of patients with involved resection margins on final histopathology, when comparing the DBT 21/  2).DCIS was the main cause of involved resection margins microscopically in 12/119 (10.1 %) of the cases with DBT and in 13/122 (10.7 %) of the cases with X-ray (Table 2).In 2/195 (1.0 %) of the cases assessed as tumor free on final histopathology (Suppl.Table 1) the patients nevertheless received a reoperation.In one case the coil was lost during primary BCS and in the other case the coil in a satellite of the index tumor was not found in the final histopathology.In 4/46 (8.7 %) of the cases, the superficial margin was involved with DCIS in the histopathology, but the surgeons considered the lesion was radically removed since the dissection was in the subcutaneous fascia.
There was no difference in the proportion of patients that received at least one re-operation between the DBT and X-ray cohorts, 16.8 % vs 19.7 %, in the adjusted analysis (p = 0.57) (Table 2).
The median total analysis time was significantly longer (p < 0.001) using intraoperative DBT (10 min) compared to X-ray (6 min) in the adjusted analysis.There was no difference in the median weight of the resected breast specimen (p = 0.57) in the adjusted analysis (Suppl.Table 2).

Diagnostic accuracy of DBT and X-ray to predict histopathological margin status
The diagnostic performance of DBT and X-ray to predict histopathological margin status is shown in Table 3.Although the sensitivity, specificity, PPV, and NPV tended to be better for DBT than for X-ray, the confidence intervals showed appreciable overlap when comparing the groups.
An illustration of a breast specimen with a TN finding on DBT is shown in (Fig. 2 b-d) and illustrated in a 2D video of the whole specimen (suppl.data).A case with a TP finding on X-ray is shown in Fig. 3 and with a FN finding on X-ray in Fig. 4. In the latter case, the radiologist assessed the intraoperative margins as tumor free, and the pathologist found it involved with DCIS in the final histopathology.The patient later received two re-operations, an additional BCS and mastectomy as a third and final surgical procedure, due to continuous DCIS involvement of the resection margins.
In 20 patients, the resected margins were difficult to assess on intraoperative imaging and the margins could not be evaluated, 8 in the DBT and 12 in the X-ray group, p = 0.38 (Suppl.Table 3).In 9 cases the patient had high mammographic breast density, in three cases the tumor border was difficult to differentiate from the surrounding fibro glandular breast tissue, in one case only a small rim of the tumor was left in the periphery of a collapsed cystic component, and in one case there was a technical problem with the imaging.There were 6 cases after NACT, three of which had complete response on breast magnetic resonance imaging (MRI) and three cases with residual tumor visible on breast MRI after NACT, where only the coil was visible on intraoperative X-ray.

Risk factors associated with at least one re-operation
We identified 5 risk factors for receiving one or more re-operation (Table 4) in the non-adjusted analysis.After adjusting for age, NACT, and grade of mammographic breast density, patients with DCIS outside  Abbreviations: n, number of specimens; DBT, digital breast tomosynthesis; TP, true positive; FP, false positive; FN, false negative; TN, true negative; CI, 95 % confidence interval; PPV, positive predictive value; NPV, negative predictive Value.n, total number of specimens that was assessed by the radiologist with the allocated imaging method.*Therewere 8 missing out of 119 specimens allocated to DBT and **12 out of 122 specimens allocated to the X-ray method, where the resected margins could not be assessed on imaging.the tumor showed the highest OR of 9.4 (95 % CI: 4.3-20.6,p < 0.001) (Table 5).Other factors associated with a risk of receiving at least one reoperation were: High, type D mammographic breast density (OR = 6.1, 95 % CI: 1.0-38.1,p = 0.047), non-evaluable tumor margins in the breast specimen on intraoperative imaging with DBT or X-ray (OR = 3.8, 95 % CI: 1.3-10.8,p = 0.016), NACT (OR = 3.0, 95 % CI: 1.0-8.8,p = 0.048), and large (T2) vs small (T1) tumor size on ultrasound (OR = 2.6, 95 % CI: 1.0-6.4,p = 0.045).

Discussion
Involved resection margins after BCS remains a clinical challenge, which most often requires a re-operation to ensure adequate removal [30,31] and to reduce the risk of local recurrence [32,33].
We found no difference in the percentage of patients with reoperation due to involved margins after BCS comparing the use of intraoperative DBT and X-ray.Previously, two retrospective studies have evaluated the impact of intraoperative DBT and X-ray on the number of involved resection margins and re-operations for patients planned for BCS, with divergent results.In concordance with our study, the most recent retrospective study with 228 invasive breast cancer patients found no difference in the percentage of patients with involved resection margins between DBT 15.8 % and X-ray method 23.9 % (p = 0.22) [34].A second retrospective study, where 191 received DBT and 466 X-ray, concluded that intraoperative DBT reduced the proportion of re-operations to 5 % as compared to 11 % with X-ray (p = 0.02) [35].However, the groups were not fully comparable in that study, since the X-ray group had more patients with DCIS, which in previous reports have been associated with an increased risk of involved histopathological margins [36,37] and thus will result in a higher proportion of re-operations.
In the present study, no significant difference was found in the diagnostic accuracy of intraoperative DBT and X-ray to predict histopathological margin status.Our findings contrast with two prospective studies that found a higher accuracy in predicting histopathological margin status for DBT of (82%-92 %) compared to X-ray (65%-79 %) [23,24].However, these studies did not explore if intraoperative DBT versus X-ray had an impact on the percentage of patients with involved histopathological margins and their surgical outcomes.We identified 5 risk factors for receiving at least one re-operation, where DCIS outside the tumor was the strongest predictor for a re-operation in consensus with two previous, large studies [33,38].The relative high percentage of re-operations of 18 % in our study can partly be explained by DCIS outside the tumor that can be difficult to visualize with both DBT and X-ray, as the suspected microcalcifications that represents DCIS are not present in all the cases [40].Furthermore, it can be difficult to differentiate between benign and suspected microcalcifications as in DCIS [16].
In most of the non-evaluable cases, the patients had a high breast density, where the radiologist could not assess the tumor margins, compromising the distinction between neoplastic and benign breast tissue.In accordance with the literature, we found that patients with extremely high mammographic breast density, after NACT, and a large (T2) tumor size have a high risk for receiving a re-operation after BCS [38][39][40].
The total analysis time of DBT in our study was 4 min longer than of X-ray.A previous report found a prolonged total surgical time of 26 min [34].Although the authors did not measure the total analysis time of each method, they suggested that the prolonged surgery time was due to a longer analysis time analyzing multiple images with DBT compared to a single X-ray image as in accordance with our findings.
In our study, DBT did not improve intraoperative margin assessment compared to X-ray.However, other promising intraoperative methods have arrived on the scene in the meantime.One of these is ultra-highresolution optical coherence tomography (UHR-OCT) combined with deep learning with a sensitivity of 93 % and a specificity of 95 % to predict histopathological margin status [41].The UHR-OCT seems to be able to differentiate cancer cells from fibro glandular and fatty tissue to a depth of 2 mm [42].Another method, intraoperative MRI of the specimen reported a sensitivity and a specificity to predict histopathological margin status of 80 % and 84 % [43].In a randomized controlled trial, use of cavity shaved margins surgical technique reduced the percentage of re-operations from 21 % to 10 % (p = 0.02) [44].This method is, however, associated with a great variation in the size of the shaved cavity samples.Finally, using the pathological assisted intraoperative gross evaluation of the specimen, combining both palpation and inspection of the fresh breast specimen showed a sensitivity of 49 %, specificity of 86 % to predict histopathological margin status [45].Despite the various promising methods, an intraoperative method that  can better visualize the DCIS in the resected margins is still needed.This is the first relatively large, randomized study to evaluate the proportion of involved histopathological margins and re-operations comparing intraoperative DBT with X-ray of the breast specimen.The study has some limitations.To comply with the routine diagnostic workflow at our departments, blinding of the surgeons and the radiologists was not an option, as well as evaluating the interobserver variability between the different breast radiologists.However, we do not believe that these limitations affected the proportion of involved histopathological margins and surgical outcomes in our study.
In conclusion, we found no difference in the number of re-operations or in diagnostic accuracy to predict histopathological margin status comparing intraoperative DBT with X-ray for intraoperative margin evaluation.DCIS outside the tumor carry the highest risk for receiving at least one re-operation.Intraoperative imaging methods that can better visualize DCIS are needed to obtain tumor free resection margins in BCS.

Fig. 3 .Fig. 4 .
Fig. 3. Intraoperative X-ray image of a breast cancer specimen with a true positive finding.a) An X-ray image from a patient with an invasive ductal cancer in the left breast with a true positive finding.The tumor was non-palpable and coil-marked by the radiologist preoperatively (arrowhead).The radiologist concluded the resection margin at 11 o'clock was involved with tumor and microcalcifications (arrow).The surgeon made an intraoperative re-resection at 9-12 o'clock.b) The finding was confirmed on final histopathology with ductal carcinoma in situ (arrowhead), outside the invasive tumor area, less than 2 mm from the re-resection margin (yellow ink) (Magnification × 50).(For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) This work was supported by following funds: Novo Nordisk Foundation [NNF19OC0057928]; Jascha Fonden [7721], Denmark; Helsefonden [19-B-0017], Denmark; Riisfort Fonden, Denmark; Danish Cancer Society [R231-A13754], Denmark and Louis-Hansen's Fonden [19-2B-5418], Denmark.The funds of this study were not involved in the design of the study, collection, management, or data analysis, in the interpretation of the results, the preparation of the manuscript or in the decision to submit the article for publication.CRediT authorship contribution statement Irina Palimaru Manhoobi: Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Writing -original draft, Writing -review & editing.Trine Tramm: Methodology, Supervision, Validation, Writing -review & editing.Søren Redsted: Methodology, Supervision, Validation, Writing -review & editing.Anne Bodilsen: Formal analysis, Supervision, Validation, Writing -review & editing.Leslie Foldager: Formal analysis, Supervision, Validation, Writing -review & editing.Peer Christiansen: Conceptualization, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Writing -review & editing.

Table 1
Perioperative characteristics of all 241 invasive breast cancer patients.
*There was one missing allocated to DBT, where the tumor was not visible on preoperative ultrasound.A t-test was performed on log transformed data for preoperative tumor size ultrasound and on log(y+1) for postoperative tumor size histology, where two patients from the X-ray group had a tumor size 0 mm after neoadjuvant chemotherapy.**Malignancy grade data was missing in 3 patients that received neoadjuvant chemotherapy, one in the DBT-and two in the X-ray group.***Special types of carcinoma (squamous, micropapillary, apocrine breast carcinoma and one no further specified).

Table 2
Final histopathological margin status and surgical outcomes.
Abbreviations: DBT, digital breast tomosynthesis; n, number; DCIS, ductal carcinoma in situ; *p-value adjusted for neoadjuvant chemotherapy, age and breast density on preoperative mammography; a) logistic regression and b) ordinal logistic regression.Intraoperative re-resection of the operating cavity was guided by the intraoperative imaging method, DBT or X-ray of the specimen.**there were 20 missing data (8 allocated to DBT and 12 to X-ray) where the resected margins could not be assessed on imaging.

Table 3
Diagnostic accuracy of intraoperative digital breast tomosynthesis and X-ray of the main breast specimen to predict histopathological resection margin status.

Table 4
Risk factors for receiving ≥1 re-operation for all 241 patients in the non-adjusted analysis.

Table 5
Risk of ≥1 re-operation in the *non-adjusted and **adjusted analysis for all 241 patients.Abbreviations: NACT, neoadjuvant chemotherapy; OR, odds ratio; CI, confidence interval; DBT, digital breast tomosynthesis; T1 and T2, Tumor stage.*Non adjusted logistic regression analysis.**Logistic regression analysis adjusted for NACT, age and preoperative breast density on mammography.***Comparisons (CI and p-value) against group A are adjusted for multiple testing by Bonferroni correction for 3 tests.